RESUMEN
A major sanctuary site for HIV infection is the gut-associated lymphoid tissue (GALT). The α4ß7 integrin gut homing receptor is a promising therapeutic target for the virus reservoir because it leads to migration of infected cells to the GALT and facilitates HIV infection. Here, we developed a core-shell nanoparticle incorporating the α4ß7 monoclonal antibody (mAb) as a dual-functional ligand for selectively targeting a protease inhibitor (PI) to gut-homing T cells in the GALT while simultaneously blocking HIV infection. Our nanoparticles significantly reduced cytotoxicity of the PI and enhanced its in vitro antiviral activity in combination with α4ß7 mAb. We demonstrate targeting function of our nanocarriers in a human T cell line and primary cells isolated from macaque ileum, and observed higher in vivo biodistribution to the murine small intestines where they accumulate in α4ß7+ cells. Our LCNP shows the potential to co-deliver ARVs and mAbs for eradicating HIV reservoirs.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Integrinas/inmunología , Intestino Delgado/efectos de los fármacos , Nanopartículas/administración & dosificación , Inhibidores de Proteasas/química , Linfocitos T/efectos de los fármacos , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/inmunología , Humanos , Íleon/efectos de los fármacos , Íleon/inmunología , Íleon/virología , Intestino Delgado/inmunología , Intestino Delgado/virología , Macaca mulatta , Ratones , Nanopartículas/química , Piridinas/administración & dosificación , Piridinas/química , Piridinas/farmacología , Pironas/administración & dosificación , Pironas/química , Pironas/farmacología , Sulfonamidas , Linfocitos T/inmunología , Linfocitos T/virologíaRESUMEN
Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021).
Asunto(s)
Citometría de Flujo/métodos , Leucocitos Mononucleares , Monitorización Inmunológica/métodos , Análisis de la Célula Individual/métodos , Biología Computacional , Humanos , Leucocitos Mononucleares/química , Leucocitos Mononucleares/clasificación , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Análisis de Secuencia de ARNRESUMEN
Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.
RESUMEN
SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).